The metabolism of the anticonvulsant drug mephenytoin exhibits a genetic polymorphism in humans. This polymorphism exhibits marked racial heterogeneity, with the poor metabolizer PM phenotype representing 13-23% of oriental populations, but only 2-5% of Caucasian populations. Two defective CYP2C19 alleles (CYP2C19*2 and CYP2C19*3) have been described, which account for more than 99% of Oriental poor metabolizer alleles but only approximately 87% of Caucasian poor metabolizer alleles. Therefore, additional defects presumably contribute to the poor metabolizer in Caucasians. Recent studies have found a third mutation CYP2C19*4, which accounts for approximately 3% of Caucasian poor metabolizer alleles. A fourth rare mutation (CYP2C19*5A) (C99,A991,Ile331;C1297T,Arg433-->Trp) resulting in an Arg433 to Trp substitution in the heme-binding region has been reported in a single Chinese poor metaboliser outlier belonging to the Bai ethnic group. The present study identifies a second variant allele CYP2C19*5B (C99-->T; A991-->G, Ile331-->Val; C1297-T, Arg433-->Trp in one of 37 Caucasian poor metabolizers. The frequency of the CYP2C19*5 alleles is low in Chinese (approximately 0.25% in the Bai ethnic group) and Caucasians (< 0.9%). However, these alleles contribute to the poor metabolizer phenotype in both ethnic groups and increases the sensitivity of the genetic tests for identifying defective alleles to approximately 100% in Chinese poor metabolizers and 92% in Caucasian poor metabolizers genotyped in our laboratory. The Arg433 to Trp mutation in the heme-binding region essentially abolishes activity of recombinant CYP2C19*5A toward S-mephenytoin and tolbutamide, which is consistent with the conclusion that CYP2C19*5 represents poor metabolizer alleles.